Extended Data Fig. 2: Decreased RPL41 mRNA staining in axons from BORCS5-KO i3Neurons. | Nature Neuroscience

Extended Data Fig. 2: Decreased RPL41 mRNA staining in axons from BORCS5-KO i3Neurons.

From: Messenger RNA transport on lysosomal vesicles maintains axonal mitochondrial homeostasis and prevents axonal degeneration

Extended Data Fig. 2

a, i3Neurons [WT, BORCS5 KO, BORCS5 KO rescued with WT BORCS5, and BORCS5 KO rescued with a G2A, myristoylation-defective BORCS5 (ΔMyr)] were immunostained for endogenous MAP2 (soma and dendrites) (magenta), neurofilament heavy chain (NFH) (axons, blue), LAMP1 (lysosomes) or LAMTOR4 (lysosomes) (all grayscale). Nuclei were stained with DAPI (yellow). Scale bars: 20 μm. Experiments were repeated three times. b, Quantification of the number of axonal puncta staining for LAMP1 or LAMTOR4 in the indicated i3Neuron lines relative to WT i3Neurons (defined as 1.0) from three experiments such as that shown in panel a. Results are represented as SuperPlots, showing the individual data points and mean of the means ± SD. Statistical significance was calculated by one-way ANOVA with Dunnett’s multiple comparisons test. Significance relative to WT: LAMP1, BORCS5 KO ***P < 0.001, BORCS5 rescue P = 0.586, BORCS5 ΔMyr rescue ***P < 0.001. LAMTOR4, BORCS5 KO ***P < 0.001, BORCS5 rescue P = 0.382, BORCS5 ΔMyr rescue ***P < 0.001. ns: not significant. Notice the depletion of lysosome-related vesicles from the axon of BORCS5-KO neurons and the rescue of this phenotype by WT but not ΔMyr BORCS5. c, The same i3Neuron lines shown in panel a were grown on coverslips for 20 days, fixed and processed for RNAscope in situ hybridization using a probe for RPL41 mRNA (white dots). Cells were also immunostained for NFH (axons, blue). Scale bars: 20 μm. Experiments were repeated three times. d, Quantification of the number of RPL41 dots per axon unit area in the indicated i3Neuron lines relative to WT i3Neurons (defined as 1.0) from three experiments such as that shown in panel c. Results are represented as SuperPlots, showing the individual data points and mean of the means ± SD. Statistical significance was calculated by one-way ANOVA with Dunnett’s multiple comparisons test. Significance relative to WT: RPL41, BORCS5 KO ***P < 0.001, BORCS5 rescue P = 0.780, BORCS5 ΔMyr rescue ***P < 0.001. Notice the depletion of RPL41 mRNA in the axon of BORCS5-KO neurons, and the rescue of this phenotype by WT but not ΔMyr BORCS5, paralleling the results for lysosome-related vesicles.

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